Parkinson's disease (“PD”) is the second most prevalent chronic neurodegenerative disease, affecting 1-3% of individuals 65 years and older. Diagnosis of PD is based on classical motor symptoms including resting tremor, rigidity, bradykinesia and postural instability. By the time PD is clinically diagnosed 60-80% of the neurons in the substantia niagra pars compacta have died leading to irreversible brain damage. Approximately 5% of the incidences of PD are familial, whereas the majority of the cases are idiopathic most likely caused by exposure to environmental factors and genetic susceptibility. Some environmental causes of PD are known; they include pesticides, fungicides, heavy metals, MPTP and traumatic brain injury. Thus, early detection of PD would be beneficial so that counseling with regards to avoidance of neurotoxins could be initiated and therapeutic intervention may be offered to patients. In addition agents that are promising as being neuroprotective for PD, including but not limited to creatine, rasagiline and minocycline, which are currently in Phase III trials, may in the future be prescribed for high risk individuals. Therefore, the identification of accurate and sensitive minimally invasive molecular biomarkers that could be used to identify PD patients would be valuable.
The loss of nigrostriatal dopamine (“DA”) neurons in PD is reflected in changes in gene expression within postmortem brains. Genes related to dopamine neurotransmission, synaptic function, electron transport, ubiquitin-proteasomal system, cytoskeletal maintenance, cell cycle and adhesion are dysregulated in PD patients (1-3). Brain tissue, however, is not a useful source for PD biomarkers. In contrast, blood biomarkers would be very useful since they can be easily obtained. In this regard, it is clear that the immune system responds to changes in DA (4). This is not unexpected since catecholamines are synthesized from tyrosine in lymphocytes and macrophages (5). In addition, dopamine receptors are expressed in low abundance in T lymphocytes and monocytes, moderately expressed in neutrophils and eosinophils, and abundantly expressed in B cells and natural killer cells (6) and DA transporters are expressed in lymphocytes (7). DA receptors are elevated and DA is reduced in the blood of PD patients compared to age-matched controls (8, 9). DA also affects the activity of regulatory T cells (10). Recently a “brain-to-T cell” pathway was proposed to explain how peripheral T lymphocytes might respond to DA in the brain based on the fact that T lymphoblast can cross the blood-brain barrier (11). It is not clear whether the changes in gene expression in the blood play a role in the etiology or progression of PD, but one intriguing study showed that CD4+ lymphocytes infiltrated the postmortem brains of PD patients (12). In addition, using a mouse model of PD the study showed that CD4+ T cells caused cell-mediated dopaminergic toxicity that lead to neurodegeneration (12). Thus, identification of changes in gene expression in the blood may provide beneficial information with regard to the cause or progression of PD.
More recently, RNA biomarkers that are predictive of PD were identified in blood cells (13, 14). In one study, 22 genes that were identified that were differentially expressed in PD patients compared to controls (14). In another study, the expression of the genes SCNA, which encodes a synuclein, and the heme metabolism genes ALAS2, FECH and BLVRB were coordinately up-regulated in blood cells of PD patients compared to controls (14). In addition the expression of SCNA was induced in the substantia nigra of PD patients (14). In the original RNA biomarker study standard cDNA microarrays were used and therefore changes in the abundance of mRNAs were monitored, which is a reflection of transcription and RNA stability.
Few studies have searched for changes in pre-mRNA splicing that occur in PD. In one study differential expression of a parkin splice variant in leukocytes of PD patients was observed (15). More recently, splicing was found to be dysregulated in PD patients (16). Because having additional RNA biomarkers would be very helpful, and since most pre-mRNAs are alternatively spliced, we chose to search for splice variant specific mRNA biomarkers of PD. Most splice variant specific markers would be masked in a standard cDNA microarray study since the up-regulation of one splice variant and the down-regulation of another variant of the same pre-mRNA might cancel each other out such that the overall abundance is unchanged. Because of this, many potential splice variant-specific RNA biomarkers may have been overlooked in earlier studies. In this study, we used splice variant-specific microarrays to identify 14 mRNA biomarkers of PD.